The spermatogonial stem cell is unique among the stem cells in the adult body in that it is the only cell that divides and can contribute genes to subsequent generations. Presently, it is defined exclusively by function and no markers are available to identify a spermatogonial stem cell in vivo or in vitro. Donor-derived spermatogenesis in a recipient testis after transplantation provides the only unequivocal functional assay. The long-term goal of this project is to identify and characterize markers for spermatogonial stem cells in mammalian testes. Specific aims are to screen cDNA libraries from purified populations of gonocytes likely to share functional characteristics with the spermatogonial stem cells that derive from them for differentially expressed genes in goats and pigs as model species; and to investigate functional competence of isolated gonocytes by transplantation to recipient testes. Germ cells will be isolated by micromanipulation from testes of newborn pigs and goats based on their unique size and morphology in these species and will be used to generate cDNA libraries to identify differentially expressed genes by subtractive hybridization with libraries obtained from spermatocytes and Sertoli cells. To investigate the functional equivalence of these germ cells to spermatogonial stem cells, they will be transplanted to heterologous mouse recipients as well as homologous recipients, and recipient testes will be monitored for donor-derived spermatogenesis by immunohistochemistry and introduction of a marker gene into the donor cells. Due to the central role of spermatogonial stem cells in the male germ line, gene expression patterns likely are conserved between mammalian species and potential markers identified in these model species are expected to also be invaluable tools for the study and manipulation of spermatogenesis in laboratory animals and humans.